(A) RT-PCR expression of rFTR1 from R. oryzae mycelia incubated in iron-replete, iron chelation (deferasirox), or reversal of iron chelation (deferasirox saturated with ferric chloride) conditions. Expression of the 18S rDNA was included to verify the quality of RNA extraction. (B) Diagram demonstrating the strategy for constructing R. oryzae GFP expression vector. Promoter denotes either rFTR1p or ACT1p. (C) GFP expression in R. oryzae driven by rFTR1p or ACT1p as determined by confocal images and flow cytometry of R. oryzae grown in iron-replete medium or medium containing deferasirox alone or deferasirox saturated with ferric chloride. GFP expression was revealed by green fluorescent cells by confocal microscopy, and the percentage of fluorescent cells in channel FL1 (y axis) by flow cytometry. In contrast to GFP under the control of the ACT1p, which was constitutively expressed regardless of growth conditions, GFP under the control of the rFTR1p was expressed only in the presence of iron chelation conditions (deferasirox [Def]).

Mice infected via the tail vein were treated with placebo (hydroxypropylcellulose carrier), deferasirox, or deferasirox plus iron (FeCl₃, 10 mg/kg) to reverse the effect of iron chelation. (A) Survival of mice (n ≥ 7 per group) infected with R. oryzae 99-892 (2.2 × 10⁴ spores) and treated with various doses of deferasirox. (B) Brain and kidney fungal burden of mice (n = 11 per group) infected with R. oryzae 99-892 (4.2 × 10⁴ spores) and treated with placebo, deferasirox (10 mg/kg twice daily), or deferasirox plus iron. Organs were harvested on day 4 after mice received 3 daily treatments. Data are displayed as median ± interquartile ranges. The y axis reflects the lower limit of detection of the assay. (C) H&E-stained kidney sections of mice infected with R. oryzae 99-892 and treated with deferasirox, deferasirox plus ferric chloride, or placebo as described in B. Arrows indicate R. oryzae hyphae in tissue. Sections are representative of findings throughout organs in all mice in the respective groups. Original magnification, ×400. (D) Survival of diabetic ketoacidotic mice (n = 24 from 3 separate experiments with similar results) infected via the tail vein with R. oryzae 99-880 (average inoculum, 1.3 × 10³ spores) and 24 hours later treated with 10 mg/kg deferasirox twice daily for 7 days. *P < 0.05 for survival and ^†P < 0.002 for tissue fungal burden compared with placebo or deferasirox plus ferric chloride.

Survival of mice infected with 10⁷ spores of R. oryzae 99-880 and 24 hours later treated with placebo (hydroxypropylcellulose carrier, n = 13), deferasirox (10 mg/kg, twice daily, n = 13), or deferoxamine (50 mg/kg, n = 8). *P < 0.009 compared with placebo- or deferoxamine-treated mice; **P = 0.047 compared with placebo-treated mice.

Diabetic ketoacidotic mice were infected via the tail vein with 3.1 × 10⁴ spores of R. oryzae 99-892 and 24 hours later treated with placebo, deferasirox, or deferasirox plus ferric chloride. Mice (n = 11) were sacrificed and spleens and kidneys collected 4 days after infection. Data are presented as median ± interquartile ranges. (A) Frequencies of Th1 and Th2 splenocyte. (B) Whole-organ cytokine analysis by cytometric bead array. *P < 0.02 compared with placebo or deferasirox plus ferric chloride; **P < 0.05 and ^†P < 0.07 compared to deferasirox plus ferric chloride.

(A) Survival of diabetic ketoacidotic mice (n > 16 from 2 separate experiments with similar results) infected via the tail vein with R. oryzae 99-880 (average inoculum, 1.5 × 10³ spores) and treated with combination of deferasirox (10 mg/kg twice daily for 7 days) and LAmB (15 mg/kg for 4 days). (B) Tissue R. oryzae burden in brains and kidneys of mice (n > 7) infected via the tail vein with R. oryzae 99-880. Treatment began 24 hours after infection and consisted of placebo, deferasirox (10 mg/kg, twice daily), LAmB (15 mg/kg/d), or a combination of both drugs. Organs were harvested on day 3 after animals received 2 daily treatments. Data are presented as median ± interquartile ranges. The y axis reflects the lower limit of detection of the assay. *P < 0.003 compared with placebo; **P < 0.003 compared with placebo, deferasirox, or LAmB; ^†P < 0.01 compared with placebo or deferasirox.

Cyclophosphamide-treated mice (n = 19 from 2 separate experiments with similar results) were infected via the tail vein with 2.7 × 10³ spores of R. oryzae 99-892. Mice were treated 24 hours after infection with placebo or deferasirox (10 mg/kg) administered every day or every other day for a total of 5 doses. *P = 0.037 compared with placebo.